Zn finger homeodomain 1

Transcriptional Regulation

The mesodermal zfh-1 expression requires the products of the twist and snail genes. In twist mutants, all the early mesodermal staining with anti-ZFH-1 antibody posterior to the cephalic furrow is lost, although expression anterior to the furrow, as well as the later expression in the developing CNS is unaffected. A similar result is obtained in snail mutants (Lai, 1991).

During germ band elongation, widespread dpp expression in the dorsal ectoderm patterns the underlying mesoderm. These Dpp signals specify cardial and pericardial cell fates in the developing heart. At maximum germ band extension, dpp dorsal ectoderm expression becomes restricted to the dorsal-most or leading edge cells (LE). A second round of Dpp signaling then specifies cell shape changes in ectodermal cells leading to dorsal closure. A third round of dpp dorsal ectoderm expression initiates during germ band retraction. This round of dpp expression is also restricted to LE cells but Dpp signaling specifies the repression of the transcription factor Zfh-1 in a subset of pericardial cells in the underlying mesoderm. Surprisingly, cis-regulatory sequences that activate the third round of dpp dorsal ectoderm expression are found in the dpp disc region. The activation of this round of dpp expression is dependent upon prior Dpp signals, the signal transducer Medea, and possibly release from dTCF-mediated repression. These results demonstrate that a second round of Dpp signaling from the dorsal ectoderm to the mesoderm is required to pattern the developing heart and that this round of dpp expression may be activated by combinatorial interactions between Dpp and Wingless (Johnson, 2003).

If a second round of Dpp LE expression influences mesodermal cell fate during late stages of heart development, it would be necessary to be familiar with the wild-type expression patterns of cardial and pericardial cell markers such as Even-skipped (Eve), Seven-up, Zfh-1, and E7 3rd 63 (an enhancer trap in seven up. An unusual feature of Zfh-1 expression was noted that has not been previously described. Zfh-1 is a zinc finger and homeobox containing protein that is widely expressed in the dorsal mesoderm during early stages of heart development. Following germ band retraction, Zfh-1 becomes restricted to pericardial cells and is detected in these cells throughout the remainder of heart development. Analysis of zfh-1 loss of function mutations shows that at early stages of heart development zfh-1 is required to maintain Eve expression but that at late stages Zfh-1 and Eve are expressed in nonoverlapping sets of pericardial cells. Examination of late stage zfh-1 mutant embryos revealed morphological defects in the heart that reflect the effects of Zfh-1 on other, as yet unidentified, pericardial genes. It was noticed that the total number of pericardial cells expressing Zfh-1 decreases significantly from stage 13 to stage 17 in wild-type embryos (Johnson, 2003).

This study suggests that a second round of dpp LE expression leads to a second round of Dpp dorsal ectoderm to mesoderm signaling. The role of the second round of Dpp LE signaling appears to be the specification of a novel subset of pericardial cells by repressing the expression of the transcription factor Zfh-1. Sequences in the dpp disc region and the signal transducer Medea are required for the second round of dpp LE expression (Johnson, 2003).

Expression data reveal that a second round of dpp LE expression initiates during germ band retraction. Genetic analyses suggest that these Dpp signals influence pericardial cell fate specification by repressing Zfh-1 expression. These results provide an explanation for the observation that p-Mad is detectable in pericardial cells of germ band retracted embryos. Most likely, the second round of Dpp LE signaling stimulates p-Mad accumulation in pericardial cells during the repression of Zfh-1 expression (Johnson, 2003).

This study also extends previous studies of Zfh-1. To date, the only known function of Zfh-1 in the mesoderm is to maintain Eve expression in pericardial cells during early stages of heart development. However, late stage zfh-1 mutant embryos show heart defects not explainable by the failure to maintain Eve expression. These results led to the suggestion that Zfh-1 has additional roles during late stages of heart development. The presence of unexplained heart defects in zfh-1 mutant embryos supports the hypothesis that the second round of Dpp LE signaling functions, through repression of Zfh-1, to specify a single pericardial cell type during late stages of embryonic heart development. Perhaps the heart defects of zfh-1 mutants are due to an excess of non-Zfh-1-expressing pericardial cells (Johnson, 2003).

Two arguments are presented suggesting that the second round of Dpp dorsal ectoderm to mesoderm signaling is also conserved in vertebrate development. (1) BMP signaling represses endothelial cell fates in the cranial vasculature of zebrafish during late stages of embryonic development. Evidence for the repression of endothelial cell fate is an overabundance of endothelial cells in the cranial blood vessels of violet beauregarde (vbg) mutants. vbg encodes the BMP type I receptor similar to the Drosophila Dpp type I receptor Saxophone. The similarity of the mutant phenotypes (overabundance of Zfh-1 expressing pericardial cells in dpp^d6 mutants and overabundance of endothelial cells in vbg mutants) is intriguing. (2) SIP1 (Smad interacting protein) is most likely the vertebrate homolog of Zfh-1. Experiments in Xenopus show that SIP1 expression in the mesoderm is repressed by BMP overexpression and induced in the absence of BMP signaling. The similarity of the interactions and their tissue specificity (Dpp represses Zfh-1 in mesodermally derived pericardial cells and in BMP represses SIP1 in mesodermal cells) suggests evolutionary conservation (Johnson, 2003).

In summary, the role and regulation of a second round of dpp LE expression is described that corresponds to a second round of Dpp signaling from the dorsal ectoderm to the mesoderm during late stages of heart development. A second round of Dpp LE signaling represses the expression of the transcription factor Zfh-1 in a subset of pericardial cells. A subset of the enhancers driving this round of dpp LE expression are located in the dpp disc cis-regulatory region. Expression from these enhancers requires prior Dpp signaling and possibly release from dTCF-mediated repression by Wg. The continued analysis of the second round of Dpp LE signaling will provide new clues to understanding vertebrate cardiovascular development (Johnson, 2003).

Specification of Drosophila aCC motoneuron identity by a genetic cascade involving even-skipped, grain and zfh1

During nervous system development, combinatorial codes of regulators act to specify different neuronal subclasses. However, within any given subclass, there exists a further refinement, apparent in Drosophila and C. elegans at single-cell resolution. The mechanisms that act to specify final and unique neuronal cell fates are still unclear. In the Drosophila embryo, one well-studied motoneuron subclass, the intersegmental motor nerve (ISN), consists of seven unique motoneurons. Specification of the ISN subclass is dependent upon both even-skipped (eve) and the zfh1 zinc-finger homeobox gene. ISN motoneurons also express the GATA transcription factor Grain, and grn mutants display motor axon pathfinding defects. Although these three regulators are expressed by all ISN motoneurons, these genes act in an eve->grn->zfh1 genetic cascade unique to one of the ISN motoneurons, the aCC. The results demonstrate that the specification of a unique neuron, within a given subclass, can be governed by a unique regulatory cascade of subclass determinants (Garces, 2006).

Why do these three genes act in a unique fashion in aCC, and why is grn and zfh1 sensitive to Notch specifically in this ISN motoneuron? One explanation may be that the differential input from upstream regulators, such as Ftz, Pdm1, Hkb and Pros, acts to modify the genetic interactions between eve, grn and zfh1. Another possibility is that the relative level of each factor plays an important role in dictating different cellular fates. Studies of the related Isl1 and Isl2 LIM-homeobox genes suggest that their involvement in motoneuron subclass specification is not primarily the result of the unique activity of each gene, but rather by the combined 'generic', tightly temporally controlled, Isl1 and Isl2 levels. Similarly, the different expression levels of the transcription factor Cut have been shown to play instructive roles during the specification of neuronal cell identities within the PNS. Different levels of expression of Grn and Zfh1 have been observed; while Grn is strongly expressed in aCC and weakly in RP2, Zfh1 expression shows an opposite distribution. It is tempting to speculate that these levels may be instructive for ISN motoneuron specification (Garces, 2006).

In the VNC, mutually exclusive expression is observed between Grn and Hb9 (and Islet) in different subsets of interneurons and motoneurons. Cross-inhibitory interactions between eve and Hb9 has been shown to contribute to their mutually exclusive expression patterns, and functional studies demonstrate that eve and Hb9 regulate axonal trajectories of dorsally and ventrally projecting axons, respectively. These observations are reminiscent of the cross-repressive interactions between classes of regulators that act to determine, refine and maintain distinct progenitor domains along the dorsoventral axis of the vertebrate neural tube. eve is important for proper grn and zfh1 expression in aCC, but not in RP2. These results are consistent with previously reported observations that the requirement for eve in axonal guidance is somewhat more stringent in aCC than in RP2, leading the the proposal that there may be different target genes for Eve in these two motoneurons (Garces, 2006).

Zfh1 expression was previously shown to depend upon Notch signaling activity in the aCC/pCC sibling pair as mutations in spdo or mam, members of the Notch signaling pathway, lead to de-repression of Zfh1 in pCC. Using the same allelic combinations, de-repression of grn was also observed in the pCC. Whether or not grn is directly suppressed by the Notch pathway remains to be seen, but it is interesting to note that in vertebrates, gata2/3 have been identified as targets of Notch during the differentiation of specific hematopoietic lineages (Garces, 2006).

Within the ISN subclass, the aCC motoneuron pioneers the ISN to innervate the dorsal-most muscle, muscle 1. A number of genetic and cell-ablation studies have convincingly shown that aCC plays an instructive pioneer role and guides the follower U motoneurons along the ISN nerve. These results lend support for the proposed instructive role of aCC in ISN formation. However, these studies indicate that aCC may not be essential for ISN formation. First, using RN2-GAL4 to visualize aCC and RP2, aberrant innervation of muscle 8 were frequently found (35% of hemisegments) in grn mutants. However, an axonal projection was simultaneously observed at the vicinity of the dorsal muscles 2/10. In grn mutants, zfh1 expression is specifically lost in aCC but maintained in RP2. Given the role for zfh1 in motor axon pathfinding, it is proposed that aberrant innervation of muscle 8 in grn mutants, is caused by aCC and not by RP2, and that RP2 pathfinds normally to the muscles 2/10. If so, RP2 may function as a pioneer motoneuron for muscle 2 and project there without the aCC axon. Second, although the rescue of grn mutants using RN2-GAL4 is complete, it was found that using CQ2-GAL4 to specifically rescue U motoneurons does lead to a partial rescue (54% muscles 1/9 innervated compared with 15% in grn mutants). Thus, even in the absence of aCC pioneer function, the Us (presumably U1) can still project to the dorsal-most muscles. This is in line with previous studies showing that in eve aCC/RP2 mosaic mutants and in aCC/RP2 cell ablation experiments, there is still partial innervation of muscle 1/9 (Garces, 2006).

grn is part of an eve --> grn --> zfh1 transcriptional cascade crucial for specification of aCC motoneuron identity. However, the failure of grn to rescue eve, and of zfh1 to completely rescue grn, combined with the misexpression results, indicate additional roles for both eve and grn. These roles could be either in the regulation of other aCC determinants and/or in the regulation of genes directly involved in aCC axon pathfinding. Although there are no obvious candidates for additional aCC determinants, recent studies point to a candidate axon pathfinding gene. The Drosophila unc-5 gene encodes a netrin receptor and is expressed in subsets of neurons in the VNC. Misexpression of unc-5 is sufficient to trigger ectopic VNC exit in subsets of interneurons. Recent studies now show that unc-5 is specifically expressed in eve motoneurons, and that eve is necessary, but only partly sufficient for unc-5 expression. In line with these findings, it was found that whereas single misexpression of eve or grn in dMP2 neurons has very minor effects, co-misexpression of eve and grn can efficiently trigger dMP2 lateral axonal exit. This combinatorial effect of eve/grn occurs without apparent activation of zfh1. However, misexpression of zfh1 can also trigger dMP2 lateral exit. Thus, these genes appear to be able to act in an independent manner to trigger VNC exit, but in a highly context-dependent manner. A speculative explanation for not only the mutant and rescue results, but also these misexpression results, would be that all three regulators are needed for robust and context-independent activation of axon pathfinding genes such as, for example, unc-5 (Garces, 2006).

grn encodes a GATA Zn-finger transcription factor and is the ortholog of the closely related vertebrate gata2 and gata3 genes. In vertebrates, gata2/3 are expressed in overlapping domains in the nervous system, but relatively little is known about their function. Expression data and evidence from gene targeting suggest an involvement in neurogenesis, neuronal migration and axon projection. A role in specifying neuronal subtypes within the context of neural tube patterning is emerging and recently a role for gata2/3 during 5-HT neuron development has been reported. The role of gata3 in the development of the inner ear has been of particular interest, and in humans, mutations in this gene have been linked to HDR syndrome, which is characterized by hypoparathyroidism, deafness and renal defects. In the mouse, gata3 is expressed in auditory but not vestibular ganglion neurons during development. The mouse gata3 mutant shows auditory ganglion neuron loss and efferent nerve misrouting, revealing that gata3 regulates molecules associated with neural differentiation and guidance. These vertebrate studies, combined with the current results, suggest that gata2/3 genes, similar to other transcription factors specifying neuronal identities, such as islet1/2, evx1/2 or Hb9, and their respective orthologs in Drosophila, have maintained similar functions throughout evolution (Garces, 2006).

A Dpp signal from the dorsal ectoderm restricts the number of pericardial cells expressing the transcription factor Zfh1

During germ-band extension, Dpp signals from the dorsal ectoderm to maintain Tinman (Tin) expression in the underlying mesoderm. This signal specifies the cardiac field, and homologous genes (BMP2/4 and Nkx2.5) perform this function in mammals. A second Dpp signal from the dorsal ectoderm restricts the number of pericardial cells expressing the transcription factor Zfh1. Via Zfh1, the second Dpp signal restricts the number of Odd-skipped-expressing and the number of Tin-expressing pericardial cells. Dpp also represses Tin expression independently of Zfh1, implicating a feed-forward mechanism in the regulation of Tin pericardial cell number. In the adjacent dorsal muscles, Dpp has the opposite effect. Dpp maintains Krüppel and Even-skipped expression required for muscle development. The data show that Dpp refines the cardiac field by limiting the number of pericardial cells. This maintains the boundary between pericardial and dorsal muscle cells and defines the size of the heart. In the absence of the second Dpp signal, pericardial cells overgrow and this significantly reduces larval cardiac output. This study suggests the existence of a second round of BMP signaling in mammalian heart development and that perhaps defects in this signal play a role in congenital heart defects (Johnson, 2007).

A previous study suggested that a second round of Dpp dorsal ectoderm-to-mesoderm signaling, stimulated by enhancers located in the dpp disk region, initiates during germ-band retraction (stage 12; Johnson, 2003). This is referred to as the second round of signaling because a distinct set of enhancers located in the dpp Haplo-insufficiency (Hin) region activates Dpp dorsal ectoderm-to-mesoderm signaling during germ-band extension (stage 8). Further, the data revealed that dpp dorsal ectoderm expression driven by the Hin region enhancers persists long after germ-band retraction. These studies showed that Hin-region-driven dpp expression is sufficient for Dpp ectodermal functions such as dorsal closure and dorsal branch migration (Johnson, 2007).

Given these data, it appears that the dpp^d6 inversion prevents the augmentation of dpp expression in the dorsal ectoderm during germ-band retraction that is normally provided by disk region enhancers. The presence of numerous mesodermal phenotypes in dpp^d6 mutants (Johnson, 2003) suggests that the augmentation of dpp expression is necessary to boost Dpp dorsal ectoderm signals so that they can reach the underlying mesoderm. Perhaps there are barriers of distance or extracellular matrix density between these germ layers that must be overcome (Johnson, 2007).

The data are wholly consistent with the hypothesis that the dpp^d6 inversion prevents the augmentation of dpp expression provided by disk region enhancers during germ-band retraction. The data further suggest that the augmentation of dpp expression is necessary to boost Dpp dorsal ectoderm signals such that they can reach the underlying mesoderm. Finally, this study shown that during germ-band retraction Dpp signals maintain the boundary between pericardial cells and dorsal muscle cells via two distinct mechanisms: the regulation of gene expression and the restriction of cell proliferation. To regulate gene expression, Dpp signals directly to pericardial cells and restricts Odd and Tin expression in a zfh1-dependent manner. Dpp also limits Tin expression, independently of zfh1, by repressing the expression of mid, a stimulator of proliferation (Johnson, 2007).

With respect to zfh1-dependent regulation, the data support the hypothesis that Dpp restricts Zfh1 expression to regulate the number of pericardial cells derived solely from symmetrically dividing lineages. Lineage analyses have identified both symmetric and asymmetric cell divisions of myogenic and pericardial precursor cells. Pericardial cells are derived from four separate lineages that arise from four distinct precursor cells. Asymmetric precursor cell divisions initiating between stages 8 and 10 give rise to the Odd-positive/Seven up (Svp)-positive pericardial cells and the Eve-positive/Tin-positive pericardial cells (EPCs). In contrast, symmetric division, initiating at the same stage, establishes the Odd-positive/Svp-negative pericardial cells (OPCs) and the Tin-positive/Eve-negative pericardial cells (TPCs). dpp mutations do not affect the number of EPCs or the number of Odd-positive/Svp-positive cells. However, embryos bearing dpp mutations show an increase in the number of OPCs and TPCs. Therefore, the ectopic pericardial cells seen in dpp mutants derive from symmetrically dividing lineages (Johnson, 2007).

Previous reports have shown that regulation of asymmetric cell division is a key mechanism in establishing boundaries among the various cell types in the dorsal mesoderm. For instance, in the absence of Numb, a Notch pathway antagonist, asymmetric progenitor cell division is abrogated and the number of Odd-positive/Svp-positive cells and EPCs increases at the expense of the Svp-expressing cardial cells and Eve-expressing dorsal muscle cells, respectively. This study extends these observations by showing that pericardial cell types derived from symmetrically dividing lineages are also under strict regulatory control (Johnson, 2007).

With respect to zfh1-dependent regulation of pericardial cell number, Dpp restricts cell proliferation and, in turn, Tin expression by limiting mid expression. In wild-type embryos, cell division in the dorsal mesoderm is largely complete by the early stages of germ-band retraction (stage 11), whereas in dpp^d6 embryos cell proliferation in the dorsal mesoderm continues through stage 13. Interestingly, the number of cells expressing Zfh1 increases from stage 12 to stage 13 in wild-type embryos in the absence of cell division, demonstrating that patterning events subsequent to cell division regulate cell fate choices in the dorsal mesoderm. This hypothesis is supported by the fact that tracing pericardial cell lineages requires inducing mitotic clones by stage 8. Therefore, the ectopically dividing mesoderm cells observed in dpp^d6 embryos are derived from cells with the potential to become Tin-expressing cells (Johnson, 2007).

During stage 12, tin expression is reactivated in a subset of cardiac cells in a mid-dependent fashion, suggesting that tin expression in precursor cells alone is not sufficient for specifying the ultimate fate of their daughter cells. Moreover, misexpression of mid results in both ectopic cell division and expanded tin expression. Lineage studies support the necessity of reactivating Tin by showing that a single precursor cell gives rise to two Tin-positive/Eve-negative pericardial cells and two siblings that do not express Tin. Thus tin is not reactivated in all subpopulations of pericardial cells. The data suggest that, during stage 12, Dpp prevents tin reactivation in cells occupying lateral regions of the dorsal mesoderm by limiting mid expression (Johnson, 2007).

Development of the dorsal musculature initiates when founder cells are specified in the mesoderm. These founder cells then fuse with neighboring cells to form syncitial myofibers. In the absence of Dpp, the pericardial cell domain expands into the dorsal muscle domain and reduces expression from the dorsal muscle genes Kr and Eve. Since the separation between pericardial and dorsal muscle cells is lost in dpp mutant embryos, it is concluded that Dpp maintains the pericardial-dorsal muscle cell boundary after it is established. Moreover, reducing pericardial cell number increases Kr expression after germ-band retraction, suggesting that cross-repressive interactions between pericardial and dorsal muscle cells contribute to patterning of the dorsal mesoderm. The presence of ectopic pericardial cells in the dorsal mesoderm reduces the number of myofibers comprising the dorsal muscles even though the dorsal muscle founder cells are, for the most part, correctly specified. pMad does not accumulate in Kr-expressing founder cells yet Kr expression is significantly reduced in dpp mutant embryos. Therefore, changes in Kr and Eve expression observed in embryos with altered dpp or zfh1 activity reflect alterations in the number of myoblast fusion events in the dorsal mesoderm (Johnson, 2007).

These data extend a previous study showing that misexpressing Zfh1 reduces dMef2 expression in somatic muscles. This study demonstrates that misexpression of Zfh1 induces ectopic pericardial cells and that the presence of pericardial cells in the dorsal muscle domain reduces myoblast fusion. Therefore, reduced dMef2 expression in embryos misexpressing Zfh1 is likely the result of reduced myoblast fusion and not of direct repression of dMef2 expression by Zfh1. Further, analysis of lmd mutants that have reduced numbers of myoblasts revealed that they also contain an excessive number of pericardial cells. Together, these results suggest that maintaining the pericardial-dorsal muscle cell boundary requires Dpp-mediated cross-repressive interactions between these cell types. Thus, in the absence of Dpp, the transformation of dorsal muscle cells into pericardial cells reduces the number of myoblasts available for fusion (Johnson, 2007).

Experiments in the larvae of Drosophila and other insects suggested that pericardial cells act as nephrocytes that filter the hemolymph. These studies also showed that pericardial cells secrete proteins into the hemolymph, suggesting that one pericardial cell function may be to provide short- or long-range signals. Consistent with this, reducing pericardial cell number reduces heart rate and increases the cardiac failure rate, suggesting that pericardial cells influence the development of cardiac cells (Johnson, 2007).

This study shows that pericardial cell hyperplasia reduces the luminal distance of the heart during systole as well as diastole, resulting in an overall decrease in average pulse distance of each contraction. However, pericardial overgrowth does not alter heart rate, indicating that cardiac cells develop appropriately in the presence of ectopic pericardial cells. Luminal measurements suggest a role for pericardial cells in the mechanics of heart function. One hypothesis for this is based on the fact that pericardial cell hyperplasia results in excess levels of extracellular matrix protein Pericardin (Prc) in the extracellular matrix (ECM) surrounding the heart. Prc is a collagen IV-like ECM protein secreted at high levels from pericardial cells. In dpp mutants, excess Prc is seen predominantly in the posterior of the heart where the pulse-distance reduction was observed. It is proposed that Prc secreted by pericardial cells limits the width of the dorsal vessel at diastole and thus modulates the pulse distance of each heart contraction. Pericardial cell overgrowth would increase Prc deposition, thereby reducing the size of the diastolic heart and the pulse distance. Consistent with this hypothesis, excessive expression of ECM proteins, including collagen IV, was correlated with heart failure in patients presenting with end-stage cardiomyopathy (Johnson, 2007).

It is well documented that many of the early events driving Drosophila embryonic heart development have been conserved in vertebrates. The data provide the first basis upon which to determine if Dpp regulation of Zfh1 or Tin late in heart development is also conserved (Johnson, 2007).

Two orthologs of zfh1, Sip1 and Kheper, have been identified in vertebrates. Zebrafish embryos injected with the Dpp homolog BMP4 show reduced Kheper expression while Xenopus embryos injected with the BMP antagonist Chordin display elevated Sip1 expression. These results suggest the possibility that Dpp repression of zfh1 expression may be conserved in vertebrates. In addition, mammalian Sip1 plays an essential role in heart development. In mice, Sip1 is expressed in neural crest cells (NCCs), paraxial mesoderm, and neuroectoderm. The subset of NCCs that express Sip1 give rise to the septum and large arteries of the heart. Sip1 knockout mice fail to form these NCCs and these mice die midway through gestation with numerous heart defects. Mice lacking the BMP receptors BMPRIA or ALK2 specifically in NCCs also display numerous cardiac phenotypes. In conditional knockout of ALK2 in NCCs, abnormalities are seen in the heart's outflow tract, and conditional knockout of BMPRIA in NCCs results in heart failure and early embryonic lethality similar to Sip1 knockout mice. Thus BMP signals are required for proper specification of NCCs, and loss of BMP signaling in NCCs phenocopies Sip1 knockout mice to an extent. It is tempting to speculate that, as in Drosophila, BMP signals regulate the Zfh1 ortholog Sip1 to correctly specify NCCs and, in turn, to properly pattern the mammalian heart (Johnson, 2007).

With regard to the conservation of late-stage Dpp regulation of Tin, a recent article describing a study of mice with a conditional knockout of Nkx2.5 where expression is missing only during late stages of heart development (post E14.5) is highly relevant. Utilizing rescue of Nkx2.5 mutant embryos with BMP-signaling-pathway components, the study identified a direct connection among BMP4 signaling, Nkx2.5 activity, and heart cell proliferation. Since Nkx2.5 is the Tin homolog, BMP4 is the Dpp homolog, and the mutant phenotype (heart cell hyperplasia) is the same in both species, this suggests that this aspect of Dpp signaling is conserved in mammals. Together with this study, these results suggest that defects in late-stage BMP signaling may play a role in congenital heart defects (Johnson, 2007).

Targets of Activity

Drosophila zfh-1 is downregulated in embryos prior to myogenesis. Embryos with zfh-1 loss-of-function mutation show alterations in the number and position of embryonic somatic muscles, suggesting that zfh-1 could have a regulatory role in myogenesis. Zfh-1 is a transcription factor that binds E box sequences and acts as an active transcriptional repressor. When zfh-1 expression is maintained in the embryo beyond its normal temporal pattern of downregulation, the differentiation of somatic but not visceral muscle is blocked. One potential target of zfh-1 in somatic myogenesis could be the myogenic factor mef2. mef2 is known to be regulated by the transcription factor twist, and Zfh-1 is shown to bind to sites in the mef2 upstream regulatory region and inhibit twist transcriptional activation. Even though there is little sequence similarity in the repressor domains of vertebrate ZEB and zfh-1, evidence is presented that Zfh-1 is the functional homolog of ZEB and that the role of these proteins in myogenesis is conserved from Drosophila to mammals (Postigo, 1999c).

Among all zfh family members, Zfh-1 and ZEB share the most sequence similarity in the zinc fingers and homeodomain. The zinc fingers of ZEB bind to a subset of E boxes (and E box-like sequences), with highest affinity for the CACCTG site. The similarity in the zinc fingers of ZEB and Zfh-1 suggests that these motifs in Zfh-1 might also be DNA binding domains. Therefore, whether Zfh-1 can bind to the CACCTG site was tested. Both the N- and C-terminal zinc fingers of recombinant Zfh-1 bind to the site in gel retardation assays. This binding is abolished when the site is mutated. Furthermore, as observed for ZEB, Zfh-1 binds to only a subset of E box sequences; it fails to bind the CATTTG E box sequence. Interestingly, the Zfh-1 binding site also matches the high-affinity site recognized by the zinc finger protein Snail, and Zfh-1 binds quite efficiently to various Snail binding sites in the single-minded gene (Zfh-1 binds better than Snail to Sna5ab, the highest-affinity site). These results demonstrate for the first time that Zfh-1 is a DNA binding protein and that it shows DNA binding specificity similar to that of ZEB and Snail (Postigo, 1999c).

Both ZEB and snail are transcriptional repressors. To determine whether Zfh-1 has transcriptional activity, a reporter containing the CACCTG binding site 30 bp upstream of an enhancer was transfected in Drosophila Schneider L2 cells. These cells do not express endogenous Zfh-1 or Snail, and thus the presence of the E box site had no effect on promoter activity. However, cotransfection of a Zfh-1 or Snail expression vector results in repression. A similar level of repression by Zfh-1 was observed when the CACCTG sequence was moved 300 bp upstream of the enhancer, demonstrating that Zfh-1 (and ZEB) can repress at long range. In contrast, Snail failed to repress transcription at this long range. Expression of DNA-binding Zfh-1 (DB-Zfh-1), containing only the DNA binding domain of Zfh-1 but not the repression domain, did not repress, suggesting that the protein has separate DNA binding and repressor domains. These results demonstrate that Zfh-1, like Snail and ZEB, functions as an active transcriptional repressor when it binds to E box sequences (Postigo, 1999c).

Because of the overlap in DNA binding specificity, Zfh-1 could target the same genes as Snail. One such Snail-regulated gene is single-minded, which is normally restricted to midline cells and a subset of somatic muscle precursor cells. Snail functions to block ectopic expression of single-minded and other nonmesodermal genes in the mesoderm. Zfh-1 not only binds to the Snail sites on the single-minded promoter but also represses the activity of the single-minded promoter in transfection assays in Schneider cells even more efficiently than Snail, consistent with the finding that sites from the single-minded promoter bind to Zfh-1 more efficiently than Snail (Postigo, 1999c).

However, Snail also binds other sequences that are not shared with Zfh-1. In the rhomboid promoter, Snail sites are important to block the expression of rhomboid in the ventral regions during embryogenesis. Contrary to what was found for the Snail sites in the single-minded promoter, Zfh-1 showed little or no binding to the four Snail sites of the rhomboid promoter. And, accordingly, Zfh-1 fails to repress the transcriptional activity of the rhomboid promoter. These results demonstrate that Zfh-1 can interact with only a subset of Snail sites (Postigo, 1999c).

It is important to point out that Snail is required for zfh-1 expression and that Zfh-1 persists after Snail diminishes. Thus, the two proteins appear to be temporally distinguishable in the developing embryo. This suggests that the two proteins may regulate separate or perhaps partially overlapping sets of genes, albeit at distinct developmental stages or in distinct tissues (Postigo, 1999).

ZEB can also repress transcription in Drosophila cells. Whether Zfh-1 can repress transcription in mammalian cells was investigated. A reporter construct containing a CACCTG binding site upstream of an enhancer was used. Coexpression of DB-Zfh-1 (or DB-ZEB) does not repress the activity of the enhancer. However, transfection of an expression vector for either full-length Zfh-1 (or full-length ZEB) or DB-Zfh-1-RD-ZEB (RD-ZEB refers to the repressive domain of ZEB) does repress transcription through the binding site. Together, these results suggest that Zfh-1 also recognizes E box binding sites in mammalian cells and represses transcription when bound to these sites (Postigo, 1999c).

To determine whether Zfh-1 contains an independent repressor domain that can function when fused to a heterologous DNA binding domain, a construct was created where the region of Zfh-1 between the zinc finger domains (corresponding to the repressor domain in ZEB) was fused to the DNA binding domain of the yeast protein Gal4. Gal4-zfh-1 was tested in transfection assays with reporter plasmids containing Gal4 binding sites cloned upstream of various enhancers. Gal4-zfh-1 efficiently represses the SV40 enhancer and thymidine kinase (TK) promoter, indicating that Zfh-1 indeed contains an independent repressor domain located between the zinc finger regions (Postigo, 1999c).

The overall sequence similarity between Zfh-1 and ZEB in their repressor domains is very low. Nevertheless, when the ability of Zfh-1 to repress the activity of a number of transcription factors was tested, it was found that Zfh-1 and ZEB have similar transcription factor specificities in transfection assays. Zfh-1 is expressed in other tissues in addition to muscle (heart, gonadal cells, central nervous system), and the ability of Zfh-1 to repress various transcription factors may have a role in the regulation of gene expression in these tissues. These results indicate that Zfh-1 is an active transcriptional repressor and that the repressor domains in Zfh-1 and ZEB may be functionally similar (Postigo, 1999c).

Given the similarity between ZEB and Zfh-1 in DNA binding specificity and repressor activity, whether Zfh-1 could substitute for ZEB and block muscle differentiation in mammalian cells was tested. Transfection of myoD is sufficient to drive cells down a myogenic pathway by inducing a cascade of transcription factors including members of the myocyte enhancer family (e.g., mef2) that collaborate with myoD to amplify the muscle differentiation program. Overexpression of ZEB blocks this myogenic conversion. A construct encoding full-length Zfh-1 also efficiently blocks myogenic differentiation. As with ZEB, DB-Zfh-1 alone does not affect myogenic differentiation, even though DB-Zfh-1 binds DNA more efficiently than the full-length protein and efficiently displaces wild-type ZEB from the promoter. Therefore, Zfh-1 and ZEB do not block myogenic differentiation simply by displacing MRF proteins from the promoter; instead, their repressor domains are required for this activity. Accordingly, fusion proteins containing DB-Zfh-1 fused to RD-ZEB, DB-ZEB fused to RD-Zfh-1, or DB-Zfh-1 fused to RD-Zfh-1 also block myotube formation. These results indicate that the RD-Zfh-1 can block myogenesis in mammalian cells, suggesting that the function of zfh-1 and ZEB may be conserved from Drosophila to mammals (Postigo, 1999c).

The transfection assays in Drosophila and mammalian cells have suggested that Zfh-1 might play a negative role during muscle development in the Drosophila embryo. Loss of Zfh-1 function does not cause drastic alterations to muscle development. In zfh-1 mutant embryos, somatic and visceral muscles form and differentiate, but there are subtle defects such as loss, misplacement, and disorganization of some muscles. These results demonstrate that Zfh-1 is not required for muscle differentiation per se, but they are consistent with a regulatory role for Zfh-1 in the process. From these studies, there was no indication about its mechanism of action and whether Zfh-1 might act as a positive or negative regulator of myogenesis. Moreover, studies on the role of ZEB in muscle differentiation had been confined to in vitro assays. Therefore, the role of Zfh-1 during myogenesis in vivo was investigated (Postigo, 1999c).

Zfh-1 is downregulated prior to somatic muscle differentiation, raising the possibility that this downregulation is essential for the onset of myogenesis. While the loss-of-function phenotype appeared mild in muscle, it was of interest to see whether maintenance of Zfh-1 expression beyond the time that endogenous Zfh-1 diminishes might have a more drastic phenotype (e.g., a blocking of myogenesis as occurs in cultured cells (Postigo, 1999c).

Zfh-1 is initially expressed throughout the mesoderm, but after gastrulation it is downregulated in muscle precursors as well as most other mesodermal derivatives. Expression of Zfh-1 was maintained throughout embryogenesis by expressing the protein under control of the heat shock protein 70 promoter; muscle development was assayed by following MHC expression. First, the embryos were heat shocked at stage 9-10, which corresponds to the time that Zfh-1 is normally downregulated and is prior to MHC expression in muscle. Zfh-1 expression following heat shock was confirmed by immunohistochemistry. At stage 14, a loss of MHC expression in somatic muscles was observed; however, surprisingly MHC expression in visceral muscle appeared relatively normal. In embryos that completed embryogenesis, milder but still clear defects in MHC expression were observed in somatic muscles (Postigo, 1999c).

The embryos were also heat shocked to induce Zfh-1 expression after the onset of MHC expression (stage 12-13). In this case, little, if any, defect in MHC expression was observed in somatic muscles, indicating that once the muscles cells begin to express MHC, they are refractory to the negative effects of Zfh-1 expression. Taken together, these results are consistent with a model in which extinction of Zfh-1 expression in embryonic muscle precursors is necessary to allow muscle differentiation to proceed (Postigo, 1999c).

It was noticed that maintaining Zfh-1 expression results in a muscle differentiation phenotype similar to that seen with the loss of Mef2 (where there is a block in MHC expression in somatic muscle with less effect on visceral muscle). This phenotype is also similar to that observed when the transcriptional activator Twist is disrupted after gastrulation via a temperature-sensitive mutant. Twist is required for activation of the mef2 gene in somatic muscle, and these observations raised the possibility that Zfh-1 may act to inhibit somatic myogenesis by blocking the expression of mef2 (Postigo, 1999c).

The pattern of mef2 expression is complex and dynamic in the embryo, but mef2 expression increases in muscle precursors as they appear in the embryo. mef2 is first evident at the late cellular blastoderm stage in mesoderm primordia and continues to be expressed throughout the mesoderm during mesoderm invagination. At mid-germband extension, mef2 expression is reduced in the ventrolateral mesoderm but maintained in the dorsal region. During germband retraction, expression increases in visceral mesoderm and in somatic muscle precursors. This is around the time when Zfh-1 is downregulated. mef2 expression then increases dramatically in all somatic mesoderm, and throughout germband retraction expression continues to be high in somatic muscles (Postigo, 1999c).

Zfh-1 is also expressed in a dynamic fashion in the mesoderm, and it is downregulated in muscle precursors as they began to appear. When embryos were double immunostained for Mef2 and Zfh-1, it was found that the expression of Zfh-1 and that of Mef2 were mutually exclusive (Postigo, 1999c).

Taken together, the above results suggested that Zfh-1 might have some role in controlling the pattern of mef2 expression in muscle precursors. To test this possibility, the pattern of mef2 expression was analyzed in embryos where Zfh-1 expression is maintained by using the heat shock construct. Wild-type embryos exhibited a normal Mef2 pattern following heat shock at all stages examined. However, heat-shocked-expressing Zfh-1 embryos showed a range of defects. (1) In the most severe cases, mef2 was highly disrupted. These embryos failed to complete germband retraction and appear not to have developed far past this stage. (2) Other embryos showed a fairly normal morphology and completed embryogenesis. In these embryos, there was still clear disruption of mef2 in somatic muscle and a reduction in the number of Mef2-positive cells (stage 12). These results suggest that the downregulation of zfh-1, associated with the onset of somatic myogenesis, is required for expression of mef2 (Postigo, 1999c).

mef2 expression has been shown to depend on the existence of an enhancer element 2.3 kb upstream of the mef2 gene that is directly activated by Twist. Examination of the mef2 promoter sequence has revealed multiple Zfh-1 sites throughout the sequence that bind Zfh-1 in gel retardation assays. Do the Zfh-1 sites in the mef2 promoter block transcriptional activation by Twist? In transfection assays, it has been shown that Zfh-1 blocks transcriptional activation by Twist, and it is proposed that Zfh-1 blocks twist-mediated activation of the mef2 gene in muscle precursors until Zfh-1 expression diminishes (Postigo, 1999c).

DEVELOPMENTAL BIOLOGY

Embryonic

See the embryonic expression pattern of zfh1 at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

Antisera against Zfh-1 and Zfh-2 were used to investigate their expression patterns during embryonic development. The zfh-1 gene is expressed in the mesoderm of early embryos. ZFH-1 protein is first detected in the presumptive procephalic mesoderm. By the early stages of gastrulation, ZFH-1 protein also begins to appear in a narrow ventral band of mesodermal anlagen extending the length of the embryo. At the start of germ band elongation, ZFH-1 protein is seen in the pole cells. In late embryos ZFH-1 protein is found in a number of mesodermally-derived structures of late embryos, including the dorsal vessel, support cells of the gonads, and segment-specific arrays of adult muscle precursors. In addition, zfh-1 is expressed in the majority of identified motor neurons of the developing CNS. Identified neurons positive for ZFH-1 include RP-13, muscle pioneers, aCC (but not pCC) ventral unpaired medial, ventral intersegmental and lateral ipsisegmental neurons (Lai, 1991).

In Drosophila as well as many vertebrate systems, germ cells form extraembryonically and migrate into the embryo before navigating toward gonadal mesodermal cells. Just how the gonadal mesoderm attracts migratory germ cells is not well understood in any system. A genetic approach has been taken to identify genes required for germ cell migration in Drosophila. The role of zfh-1 is described in germ cell migration to the gonadal mesoderm. Zfh-1 protein is initially expressed in all mesodermal cells, but by stage 10, Zfh-1 levels have declined in most mesodermal cells, although high levels are maintained in extreme anterior and posterior mesodermal cells. The cells within the anterior cluster are likely to be hemocytes. During stage 10, Zfh-1-expressing mesodermal cells located at the posterior end of the embryo migrate anteriorly in two bilaterally symmetric groups between the endoderm and the interior of the dorsal mesoderm. These cells have been termed the 'caudal visceral mesoderm' as they contribute to the midgut musculature at later stages. Crocodile was used as a marker for the caudal visceral mesoderm. Croc is not expressed in the caudal visceral mesoderm in zfh-1 mutant embryos. Caudal visceral mesoderm is in close proximity to migratory germ cells during late stage 10. In zfh-1 mutant embryos, the initial association of germ cells with their final destination, gonadal mesoderm made up of the somatic gonadal precursors (SGP), is blocked. Instead, some germ cells remain attached to the gut, leading to a cluster of germ cells in the middle of the embryo during later stages of development (Broihier, 1998).

The zfh-2 gene displays a more limited expression pattern, largely restricted to the CNS of late embryos. The expression patterns of zfh-1 and zfh-2 suggest that both genes may be involved in Drosophila neurogenesis and that zfh-1 may have additional functions in mesoderm development (Lai, 1991).

Subdivision and developmental fate of the head mesoderm in Drosophila

This paper defines temporal and spatial subdivisions of the embryonic head mesoderm and describes the fate of the main lineages derived from this tissue. During gastrulation, only a fraction of the head mesoderm (primary head mesoderm; PHM) invaginates as the anterior part of the ventral furrow. The PHM can be subdivided into four linearly arranged domains, based on the expression of different combinations of genetic markers (tinman, heartless, snail, serpent, mef-2, zfh-1). The anterior domain (PHMA) produces a variety of cell types, among them the neuroendocrine gland (corpus cardiacum). PHMB, forming much of the'T-bar' of the ventral furrow, migrates anteriorly and dorsally and gives rise to the dorsal pharyngeal musculature. PHMC is located behind the T-bar and forms part of the anterior endoderm, besides contributing to hemocytes. The most posterior domain, PHMD, belongs to the anterior gnathal segments and gives rise to a few somatic muscles, but also to hemocytes. The procephalic region flanking the ventral furrow also contributes to head mesoderm (secondary head mesoderm, SHM) that segregates from the surface after the ventral furrow has invaginated, indicating that gastrulation in the procephalon is much more protracted than in the trunk. This study distinguishes between an early SHM (eSHM) that is located on either side of the anterior endoderm and is the major source of hemocytes, including crystal cells. The eSHM is followed by the late SHM (lSHM), which consists of an anterior and posterior component (lSHMa, lSHMp). The lSHMa, flanking the stomodeum anteriorly and laterally, produces the visceral musculature of the esophagus, as well as a population of tinman-positive cells that is interpreted as a rudimentary cephalic aorta ('cephalic vascular rudiment'). The lSHM contributes hemocytes, as well as the nephrocytes forming the subesophageal body, also called garland cells (de Velasco, 2005).

The mesoderm is a morphologically distinct cell layer that can be recognized in early embryos of most bilaterian phyla and that gives rise to tissues interposed between ectodermal and endodermal epithelia, including muscle, connective, blood, vascular, and excretory tissue. Besides the differentiative fate of tissues derived from it, the mesoderm shares several common properties in regard to its formation during gastrulation. The anlage of the mesoderm is sandwiched in between the anlage of the endoderm and the neurectoderm. This has been documented in most detail in anamniote vertebrates, where signals from the vegetal blastomeres (the anlage of the endoderm) act on the adjacent marginal zone of the future ectoderm to induce mesoderm. Although gastrulation proceeds quite differently in arthropods from the way it does in chordates, the proximity of the mesodermal anlage to future endoderm and neurectoderm is conserved, and numerous signaling pathways and transcriptional regulators that share similar function and expression patterns in arthropods and chordates have been identified (de Velasco, 2005 and references therein).

Following gastrulation, the mesoderm is subdivided along the dorso-ventral axis into several subdivisions laid out in a distinct dorso-ventral order. In vertebrates, cells located in the dorsal part of the mesoderm anlage give rise to notochord and somites, which in turn produce muscular, skeletal, and connective tissue. Next to the somitic mesoderm is the intermediate mesoderm that will form the excretory and reproductive system. The ventral mesoderm (lateral plate) gives rise to blood, vascular system, visceral musculature, and coelomic cavity. In arthropods, fundamentally similar mesodermal subdivisions can be recognized, and similarities extend to the relative positions these domains obtain relative to each other and relative to the adjacent neurectoderm. For example, precursors of visceral muscles, vascular system, and blood are at the edge of the mesoderm facing away from the neural primordium (ventral in vertebrates, dorsal in arthropods (de Velasco, 2005 and references therein).

The subdivision of the vertebrate mesoderm into distinct longitudinal tissue columns with different fates is seen throughout the trunk and head of the embryo. However, several significant differences between the head and the trunk are immediately apparent. For example, cells derived from the anterior neurectoderm form the neural crest that migrates laterally and gives rise to many of the tissues that are produced by mesoderm in the trunk. As a result, the fates taken over by the head mesoderm are more limited than those of the trunk mesoderm. In contrast, the head mesoderm produces several unique lineages, such as the heart (cardiac mesoderm) and a population of early differentiating macrophages. Moreover, some of the signaling pathways responsible for inducing different mesodermal fates in the trunk appear to operate in a different manner in the head. A recently described example is the Wnt signal that induces somatic musculature in the trunk, but inhibits the same fate in the head (de Velasco, 2005 and references therein).

The head mesoderm of arthropods, like that of vertebrates, also appears to deviate in many ways from the trunk mesoderm. For example, specialized lineages like embryonic blood cells and nephrocytes forming the subesophageal body (also called garland cells) arise exclusively in the head. That being said, very little is known about how the arthropod head mesoderm arises and what types of tissues derive from it. The existing literature mainly uses histology, which severely limits the possibilities of following different cell types forward or backward in time. In this paper, several molecular markers have been used to initiate more detailed studies of the head mesoderm in Drosophila. The goal was to establish temporal and spatial subdivisions of the head mesoderm and, using molecular markers expressed from early stages onward, to follow the fate of the lineages derived from this embryonic tissue. Besides hemocytes and pharyngeal muscles described earlier, the head mesoderm also gives rise to several other lineages, including visceral muscle, putative vascular cells, nephrocytes, and neuroendocrine cells. The development of the head mesoderm is discussed in comparison with the trunk mesoderm and in the broader context of insect embryology (de Velasco, 2005).

The Drosophila head mesoderm, as traditionally defined, includes all mesoderm cells originating anterior to the cephalic furrow. The formation of the head mesoderm is complicated by the fact that (unlike the mesoderm of the trunk) only part of it invaginates with the ventral furrow; by far, the majority of head mesoderm cells, recognizable in a stage 10 or 11 embryo, segregate from the surface epithelium of the head after the ventral furrow has formed. Another complicating factor is that head mesoderm cells derived from different antero-posterior levels adopt very different fates, unlike the situation in the trunk where mesodermal fates within different segments along the AP axis are fairly homogenous, with obvious exceptions such as the gonadal mesoderm that is derived exclusively from a subset of abdominal segments. Using several different markers, this study has followed the origin, migration pathways, and later, fates of head mesoderm cells (de Velasco, 2005).

The anterior part of the ventral furrow, called primary head mesoderm (PHM) in the following, includes cells that will contribute to diverse tissues, including muscle, hemocytes, endoderm, and several ill-defined cell populations closely associated with the brain and neuroendocrine system. For clarification, the anterior ventral furrow will be divided into the following domains:

• Domain A (PHMA): the anterior lip of the T-bar, defined by the expression of sine oculis (so), giant (gt), and a reduced level of heartless (htl).
• Domain B (PHMB): the posterior lip of the T-bar, defined by high levels of htl and tinman (tin) expression and absence of serpent (srp).
• Domain C (PHMC): the anterior part of the procephalic ventral furrow proper, defined by the expression of srp and other endoderm markers and by low levels of htl or tin.
• Domain D (PHMD): the posterior part of the procephalic ventral furrow (anterior gnathal mesoderm), which initially resembles the trunk mesoderm in expressing high levels of htl and tin. Secondarily, much of the gnathal mesoderm switches to srp expression as it adopts the fate of blood cell precursors (de Velasco, 2005).

The anterior lip of the T-bar (PHMA) is the source of the corpus cardiacum, as well as other gt-positive cells that at least in part end up as nerve cells flanking the frontal connective and frontal ganglion. These cells continue the expression of giant throughout late embryonic development; they represent a hitherto unknown class of nonneuroblast-derived neurons (de Velasco, 2005).

The posterior lip of the T-bar (PHMB) can be followed towards later stages by its continued expression of htl. These cells, called the procephalic somatic mesoderm, form a bilateral cluster that moves dorso-anteriorly into the labrum and becomes the dorsal pharyngeal musculature. Htl expression almost disappears in these cells around late stage 11, but is reinitiated at stage 12 and stays strong until stage 14, when the dorsal pharyngeal muscles differentiate. Many of the genes expressed in the somatic musculature of the trunk and its precursors (Dmef2, beta-3-tubulin) are also expressed in the procephalic somatic mesoderm (de Velasco, 2005).

The part of the ventral furrow posteriorly adjacent to the T-bar (PHMC) expresses srp, forkhead (fkh), and other endoderm/hemocyte markers. After the ventral furrow closes in the ventral midline (stage 7/8), these cells form a compact median mass, most of which represents part of the anterior endoderm that gives rise to the midgut epithelium. Starting at around this stage, the lateral part of the hemocyte-forming 'secondary head mesoderm' ingresses in between the endoderm and the surface ectoderm. It is likely that some of the PHMC cells invaginating already with the ventral furrow, along with the cells that form the anterior endoderm, also give rise to hemocytes. Precursors of hemocytes and midgut are difficult to distinguish during and shortly after ventral furrow invagination since both express srp and other markers shared between hemocytes and midgut precursors. At around stage 9, the two populations of precursors disengage. The endoderm remains a compact mesenchyme attached to the invaginating stomodeum; hemocyte precursors move dorsally and take on the shape of expanding vertical plates interposed in between endoderm and ectoderm (de Velasco, 2005).

Domain PHMD, the short portion of the ventral furrow situated posterior to the endoderm, along with a considerable portion of the mesoderm behind the cephalic furrow, forms the mesoderm of the three gnathal segments (mandible, maxilla, labium). The gnathal mesoderm in many ways behaves like the mesoderm of thoracic and abdominal segments. It gives rise to somatic muscle (the lateral pharyngeal muscles), visceral muscle, and fat body. Unlike trunk mesoderm, gnathal mesoderm does not produce cardioblasts and pericardial cells. Instead, a large proportion of gnathal mesoderm cells, joining the anteriorly adjacent secondary procephalic mesoderm, adopt the fate of hemocytes (de Velasco, 2005).

Besides the ventral furrow, other parts of the ventral procephalon produce head mesoderm in a complex succession of delamination and ingression events. The head mesoderm that forms from outside the ventral furrow will be called 'secondary mesoderm' (SHM) in the following. Based on the time of formation and the position relative to the stomodeum, the following phases and domains of secondary head mesoderm development can be distinguished.

• The early secondary head mesoderm (eSHM) ingresses and delaminates during stages 7-8 from the ventral epithelium flanking the endoderm.
• The posterior late secondary head mesoderm (lSHMp) ingresses during stages 9-11 from a similar position as the eSHM. Its cells give rise to blood cells and nephrocytes (the subesophageal body or garland cells).
• The anterior late secondary head mesoderm cells (lSHMa) delaminate lateral and anterior to the stomodeum. Coming from within the anlagen of the esophagus and epipharynx, these cells produce esophageal visceral muscle cells, as well as a novel tin-positive cell type that is interpreted as a rudimentary cephalic vascular structure (de Velasco, 2005).

Following the obliteration of the ventral furrow at stage 8, the eSHM delaminates from the ventral surface 'meso-ectoderm' (considering that this epithelium still contains mesodermal progenitors!) flanking the endodermal mass. The eSHM forms two monolayered sheets that gradually move dorsally and posteriorly; by stage 9, the eSHM cells line the basal surface of the emerging head neuroblasts. An undefined number of primary head mesoderm cells derived from domain PHMC of the ventral furrow are mingled together with the eSHM cells. The ultimate fate of the eSHM is that of hemocytes: they express srp, followed slightly later by other blood cell markers (e.g., peroxidasin and asrij). A subset of hemocytes, called crystal cells, derive from precursors that form a morphologically conspicuous cluster at the dorsal edge of the eSHM, identifiable from early stage 10 onward by the expression of lz. The mechanism by which at least part of the eSHM delaminates is unique. Thus, it is formed by the vertically oriented division of the surface epithelium, whereby the inner daughters will become eSHMe and the outer ones ectoderm. The focus of vertical mitosis has named the procephalic domain in which it occurs 'mitotic domain #9' (de Velasco, 2005).

From late stage 9 onward, the early SHMs are followed inside the embryo by the closely adjacent posterior late SHMs. One cluster of posterior late secondary head mesoderm (lSHMp) cells delaminates from the surface epithelium flanking the posterior lip of the stomodeum; a second lSHMp cluster appears at the same stage at a slightly more posterior level. The first cluster seems to contribute to the hemocyte population; the posterior cluster gives rise to the nephrocytes forming the subesophageal body (also called garland cells; labeled by CG32094). Garland cell precursors are initially arranged as a paired cluster latero-ventrally of the esophagus primordium; subsequently, the clusters fuse in the midline and form a crescent underneath the esophagus. Garland cells are distinguished from crystal cells by their size, location, and arrangement: crystal cells are large, round cells grouped in an oblong cloud dorso-anterior to the proventriculus. Garland cells are smaller, closely attached to each other, and lie ventral of the esophagus (de Velasco, 2005).

During stages 10 and 11, cells delaminate beside and anterior to the stomodeum, originating from the anlage of the esophagus and the epipharynx (labrum). These cells, called anterior late secondary head mesoderm cells (lSHMa), can be followed by their expression of tin. Two groups can be distinguished. The tin-positive cells delaminating from the esophageal anlage (es) give rise to the visceral musculature (vm) surrounding the esophagus. These cells lose tin expression soon after their segregation, but can be recognized by other visceral mesoderm markers such as anti-Connectin. More dorsally, in the anlage of the clypeolabrum (cl) delaminate, the dorsal subpopulation of the lSHMas, which rapidly migrates posteriorly on either side and slightly dorsal of the esophagus, can be found. These cells retain expression of tin into the late embryo. They assemble into two longitudinal rows stretching alongside the roof of the esophagus primordium. During late embryogenesis, they move posteriorly along with the esophagus towards a position behind the brain commissure. Many of the tin-positive SHMs apparently undergo apoptosis: initially counting approximately 25 on either side, they decrease to 12-15 at stage 14 to finally form a single, irregular row of about 15 cells total in the late embryo. These cells come into contact with the anterior tip of the dorsal vessel. This formation of previously undescribed cells, for which the term 'procephalic vascular cells', is proposed, is interpreted as a rudiment of the head aorta, which forms a prominent part of the dorsal vessel in many insect groups (de Velasco, 2005).

On the basis of additional molecular markers, the tin-positive procephalic vascular cells are further subdivided into two populations. The first subpopulation expresses the muscle and cardioblast-specific marker Dmef2; the second type is Dmef2-negative. In the dorsal vessel of the trunk, tin-positive cells also fall into a Dmef2-positive and a Dmef2-negative population. Dmef2-positive cells of the trunk represent the cardioblasts, myoendothelial cells lining the lumen of the dorsal vessel. Dmef2-negative/tin-positive cells form a somewhat irregular double row of cells attached to the ventral wall of the dorsal vessel. The ultimate fate of these cells has not been explored yet. However, preliminary data suggest that they develop into a muscle band that runs alongside the larval dorsal vessel. This would correspond to the situation in other insects in which such a ventral cardiac muscle band has been described (de Velasco, 2005).

The role of tinman in the formation of the procephalic vascular rudiment was investigated by assaying tin-mutant embryos for the expression of Dmef2. Similar to the cardioblasts of the trunk, the Dmef2-positive cells of the procephalic vascular rudiment are absent in tin mutants. It is quite likely that the (Dmef2-negative) remainder of the procephalic vascular rudiment is affected as well by loss of tin, but in the absence of appropriate markers (besides tin itself, which is not expressed in the mutant), it was not possible to substantiate this proposal (de Velasco, 2005).

At the time of appearance of the ventral furrow, segmental markers such as hh do not allow the distinction between distinct 'preoral' segments. Thus, hh is expressed in a wide procephalic stripe in front of the regularly sized mandibular stripe. During stage 7, the procephalic hh stripe splits into an anterior, antennal stripe and a posterior, short, intercalary stripe. The anterior lip of the ventral furrow (domain PHMA) coincides with the anterior boundary of the antenno-intercalary stripe. Thus, the primary head mesoderm and endoderm originating from within the anterior ventral furrow can be considered a derivative of the antennal and intercalary segments. This interpretation is supported by the expression of the homeobox gene labial (lab) found in the intercalary segment. The labial domain covers much of the anterior ventral furrow, including domains PHMB-C (de Velasco, 2005).

Morphogenetic movements in the ventral head, associated with the closure of the ventral furrow, the formation of the stomodeal placode, and the subsequent invagination of the stomodeum result in a shift of head segmental boundaries. The antennal segment tilts backward, as can be seen from the orientation of the antennal hh stripe that from stage 8 onward forms an almost horizontal line, connecting the cephalic furrow with the sides of the stomodeal invagination (which falls within the ventral realm of the antennal segment, in Drosophila as well as other insects). Since the expression of hh, like that of engrailed (en), coincides with the posterior boundary of a segment, the territory located ventral to the antennal hh stripe falls within the intercalary segment. This implies that most, if not all, of the posterior late SHM, is intercalary in origin. It is further plausible to consider that the anterior lSHM belongs to the intercalary and antennal segment. The vascular cells of the head, a conspicuous derivative of the anterior lSHM in Drosophila, are derived from the antennal mesoderm in other insects. The labrum, with which much of the anterior lSHM is associated, represents a structure that has always been difficult to integrate in the segmental organization of the head. Most likely the labrum represents part of the intercalary segment; this would help explain some of the unusual characteristics of the head mesoderm (de Velasco, 2005).

In conclusion, several fundamental similarities are found between the mesoderm of the head and that of the trunk regarding the tissues they give rise to, and possibly the signaling pathways deciding over these fates. After an initial phase of structural and molecular homogeneity, the trunk mesoderm becomes subdivided into a dorsal and a ventral domain by a Dpp-signaling event that emanates from the dorsal ectoderm. The dorsal domain, characterized by the Dpp-dependent continued expression of tinman, becomes the source of visceral and cardiogenic mesoderm, among other cell types. A role of Dpp/BMP signaling in cardiogenesis seems to be conserved among insects and vertebrates. Subsequent signaling steps, involving both Wingless and Notch/Delta, separate between these two fates and further subdivide the cardiogenic mesoderm into several distinct lineages, such as cardioblast, pericardial cells, and secondary hemocyte precursors (lymph gland). As a result of these signaling events, Tinman and several other fate-determining transcription factors become restricted to their respective lineages: tin to the cardioblasts, odd to pericardial cells and hemocyte precursors, zfh1 and srp to hemocyte precursors and fat body. Dmef2 and several other transcription factors become restricted to various combinations of muscle types (somatic, visceral, cardiac) (de Velasco, 2005).

In the head mesoderm, the above genes are associated with similar fates. Tin and Dmef2 appear widely in the procephalic ventral furrow and the anterior lSHM before getting restricted to the procephalic vascular rudiment and/or the pharyngeal musculature, respectively. In contrast with the initially ubiquitous expression of Tin and Dmef2 in the trunk mesoderm, those parts of the head mesoderm giving rise to hemocytes (PHMC, posterior lSHM) never express these mesodermal genes. Previous work has shown that the head gap gene buttonhead (btd) is responsible for the early repression of tin in the above mentioned domains of the head mesoderm. The early absence of Tin and Dmef2 in the head mesodermal hemocyte precursors is paralleled by the presence of Srp and Zfh1 in these cells. Interestingly, Srp/Zfh-positive cells of the head produce only hemocytes and no fat body, suggesting that an as-yet-uncharacterized signaling step prevents the formation of fat body in the head. It is tempting to speculate that there exists within the mesoderm a 'blood/fat body equivalence group'. Blood cells and fat body share not only the expression of fate-determining genes such as srp and zfh1, but also, later, functional properties that have to do with immunity. In the trunk, the blood/fat body equivalence group gives rise mostly to fat body, producing only a limited number of hemocyte precursors in the dorsal mesoderm of the thoracic segments. In the head, on the other hand, all cells of the equivalence group become hemocytes (de Velasco, 2005).

Attention is drawn to another mesodermal lineage that produces related, yet not identical, cell types in the trunk and the head: the nephrocytes. Nephrocytes are defined by their characteristic ultrastructure (membrane invaginations sealed off by junctions) that attests to their excretory function. In the trunk, nephrocytes are represented by the pericardial cells that settle beside the cardioblasts; a newly discovered nephrocyte population ('star cells') invading the Malpighian tubules is derived from the mesoderm of the tail segments. In the head, nephrocytes aggregate near the junction between esophagus and proventriculus as the subesophageal body, also called garland cells. The fact that from the early stages of development onward different transcription factors are expressed in garland cells and pericardial cells suggests that these cells perform similar, yet not fully overlapping, functions (de Velasco, 2005).

Effects of Mutation or Deletion

Ectopic expression of the zfh-1 gene produces defects in the CNS. Adult survivors of both larval and pupal overexpression often have missing, misplaced, and duplicated thoracic macrochaetae. Roughened eyes are produced by ectopic expression during late larval development. The eye roughening is characterized by disordered, irregularly sized facets and is most pronounced in posterior eye regions (Lai, 1991).

Phenotypic analysis of zfh-1 mutant embryos reveals that the gene is not required for the initial segregation of the mesoderm or for the differentiation of mesodermally derived tissues. Rather, loss of zfh-1 function results in various degrees of local errors in cell fate or positioning. The ventral-oblique and the dorsal muscles are usually the most severely affected. The ventral-longitudinal and pleural muscles appear fairly normal in number and attachment but lack the taut straight appearance of the wild type. There are a variety of errors in segregation of muscle precursors. Mutants have missing muscles, misplaced muscles, and nuclei within a muscle are disorganized and spread further from the cluster than normal. The foregut and hindgut appear normal, but the midgut is abnormal. Constrictions start to form but rarely complete the subdivision of the yolk, and the elongation and narrowing of the gut occurs in a partial and very uneven fashion. In some mutants there is a prononunced kink in the heart. In others, there are breaks in anterior parts of the heart: the two sides fail to come together for part of the length of the heart or cells fail to join to make a continuous tube lengthwise. Gonads are abnormal: the normally compact structure of the gonads appears to be more dispersed. A range of defects are seen in pole cells. Adult muscle precursors are missing or displaced (Lai, 1993).

In many animal groups, an interaction between germ and somatic lines is required for germ-line development. In Drosophila, the germ-line precursors (pole cells), which form at the posterior tip of the embryo migrate toward the mesodermal layer where they adhere to the dorsolateral mesoderm, which ensheaths the pole cells to form the embryonic gonads. These mesodermal cells may control the expression of genes that function in the development of germ cells from pole cells. However, such downstream genes have not been isolated. In this study, a novel transcript, indora(idr), is identified that is expressed only in pole cells within the gonads. The nucleotide sequence of the 1.5 kb cDNA predicts a protein of 131 amino acids. The amino acid sequence shows no significant homology to any known proteins. The putative Idr protein is highly basic (calculated isoelectric pH is 10.1). During normal development, the expression of idr transcripts become discernible in pole cells at the embryonic stage 14, when pole cells are incorporated into the gonads. Expression persisted in pole cells until the completion of embryonic development. idr expression is undetectable in the adult germ line. However, the possibility that a trace amount of IDR mRNAs is expressed in somatic cells as well as in the germ line throughout most of the life cycle cannot be excluded, because Northern blot analysis reveals that idr transcripts are detectable from late embryogenesis to adulthood (Mukai, 1998).

Reduction of idr transcripts by an antisense idr expression causes the failure of pole cells to produce functional germ cells in females. Furthermore, idr expression depends on the presence of the dorsolateral mesoderm, but it does not necessarily require its specification as the gonadal mesoderm. In order to determine the source of the mesodermal cue, idr expression was analyzed in the absence of the mesodermal cells that make up the gonads. The origin and development of the somatic components of the gonads are described. The somatic gonad precursors (SGPs) are specified from the dorsolateral mesoderm within PS 10-12 at stage 11. In tin;zfh-1 double-mutants, no dorsolateral mesoderm is formed, which results in loss of SGPs. In these embryos, pole cells pass through the midgut epithelium, but subsequently they are dispersed around the midgut. idr expression is drastically reduced in tin;zfh-1 double-mutants. This result shows the requirement of the dorsolateral mesoderm for idr expression in pole cells. It was next asked whether the specification of the dorsolateral mesoderm as SGPs is needed to induce idr expression in pole cells. To examine this, abd-A and iab-4 mutations were used. abd-A function is required in the mesodermal cells for the specification of SGPs. In abd-A mutant embryos, pole cells pass through the midgut wall and are normally associated with the dorsolateral mesoderm. However, they do not coalesce with the pole cells to form the gonads due to their failure to be specified as SGPs. Consequently, pole cells are released from the mesoderm and scattered throughout the embryo. In these embryos, the dispersed pole cells express idr during stages 14-16. Furthermore, a regulatory mutation in the abd-A locus, iab-4, also has no deleterious effect on idr expression. Thus, the specification of the dorsolateral mesoderm as SGPs is dispensable for idr expression. These findings suggest that the induction of idr in pole cells by the mesodermal cells is required for germ-line development (Mukai, 1998).

The Drosophila Brachyury homolog brachyenteron (byn) is essential for the development of hindgut, anal pads and Malpighian tubules. byn is activated by the terminal gap gene tailless (tll) in a region of 0%-20% egg length of the syncytium (0% = posterior tip). With completion of cellularization, the byn expression becomes downregulated in the posteriormost cap of the embryo, which will later form the posterior midgut, by the terminal gap gene huckebein (hkb). Thus, the expression of byn is confined to a ring of cells from about 10%-20% egg length. The dorsal and the lateral aspects of that ring correspond to the proctodeum, from which the hindgut, the anal pads and the Malpighian tubules later develop. Intriguingly, hkb also determines the posterior extent of the ventral mesoderm primordium by repressing the mesodermal determinant snail (sna). This suggests that the ventralmost aspect of byn expression might comprise the posterior tip of the mesoderm primordium (Kusch, 1999).

The visceral musculature of the larval midgut of Drosophila has a lattice-type structure and consists of an inner stratum of circular fibers and an outer stratum of longitudinal fibers. The longitudinal fibers originate from the posterior tip of the mesoderm anlage, which has been termed the caudal visceral mesoderm (CVM). The CVM migrates in an orderly movement anteriorly and eventually forms an outer layer of longitudinal muscle fibers surrounding the midgut. The progenitors of a second tissue, the inner sheet of circular muscles of the midgut, are recruited from 11 parasegmentally arranged clusters of dorsal mesoderm in the trunk region and are therefore referred to as trunk visceral mesoderm (TVM) (Kusch, 1999).

In this study, the specification of the CVM has been investigated and particularly the role of the Drosophila Brachyury-homolog brachyenteron. Supported by fork head, brachyenteron mediates the early specification of the CVM along with zinc-finger homeodomain protein-1. This is the first function described for brachyenteron or fork head in the mesoderm of Drosophila. The mode of cooperation resembles the interaction of the Xenopus homologs Xbra and Pintallavis. Another function of brachyenteron is to establish the surface properties of the CVM cells, which are essential for their orderly migration along the trunk-derived visceral mesoderm. During this movement, the CVM cells, under the control of brachyenteron, induce the formation of one muscle/pericardial precursor cell in each parasegment. It is here proposed that the functions of brachyenteron in mesodermal development of Drosophila are comparable to the roles of the vertebrate Brachyury genes during gastrulation (Kusch, 1999).

During germband retraction and midgut closure, the progenitors of the the outer, longitudinally oriented fibers of the visceral mesoderm, the CVM, perform an ordered movement that can be subdivided into three phases. The first migratory phase starts at early germband retraction when the cells begin to move anteriorly from their position at the posterior tip of the mesodermal germ layer and split into two tightly packed, bilaterally symmetrical clusters on each side of the posterior midgut primordium. When these clusters have reached the anterior tip of the posterior midgut primordium, the cells detach from each other and disperse anteriorly as two rows along the germband, the second phase of the migration. During this movement, the cells are arranged along the dorsal and ventral edge of the midgut primordia and are in close contact with the band of progenitors of the circular muscle fibers. The band seems to serve as a migration substratum. During the last phase of the migration, which takes place as the midgut encloses the yolk, the progenitors of the longitudinal muscle fibers spread regularly over the underlying circular muscle fibers. The cells acquire a spindle shape, then stretch in an anteroposterior direction and form about 16-20 regularly spaced longitudinal muscle fibers. These fibers reach from the proventriculus to the midgut-hindgut transition where the ureters of the Malpighian tubules insert. The foregut and the hindgut lack any longitudinal muscles and are solely covered by the inner layer of circular muscles (Kusch, 1999).

The specification of the CVM and its fate were monitored by the detection of Byn protein or the expression of CVM-specific markers like croc-lacZ and cpo-lacZ. The initial byn expression at the posterior pole is regulated by tll and hkb. Thus it is likely that the CVM cells are specified under the control of the same genes. In fact, in hkb embryos, the size of the CVM primordium is enlarged and comprises more cells than normal. This corroborates the notion that the CVM primordium constitutes the most posteriorly located mesoderm primordium. tll expression reaches more anteriorly than the hkb domain and encompasses the primordia of the proctodeum and of the CVM. One would therefore expect that the formation of the CVM is entirely dependent on tll. Indeed, this is the case: the CVM is missing in tll mutant embryos. Part of the function of tll seems to be mediated by byn. In byn mutants, a significantly reduced number of CVM cells is seen, and these few cells form clusters that are less compact and migrate significantly slower than in wild type. Later, they fail to contact the TVM and do not distribute along the germband. During stage 11, most of the cells acquire a condensed appearance resembling apoptotic bodies. A high level of apoptosis is detected in the proctodeum of byn embryos as well as in the posteriormost mesoderm. By stage 13, cells with the properties of the CVM are not detectable any longer in the mutants and, as expected from this, the dissected midguts of byn embryos lack the outer, longitudinal muscle fibers (Kusch, 1999).

Only the anterior and the posterior mesoderm are competent to be specified by byn as CVM, in conjunction with fkh. Therefore, at least one other gene must exist that confines the competence to form CVM to these two regions. A good candidate for this gene is zinc finger homeodomain protein-1 (zfh-1). At the blastoderm stage, zfh-1 is expressed in high levels in the terminal regions of the mesoderm including the primordium of the CVM. zfh-1 is essential for the migration of the CVM: in zfh-1 mutant embryos, CVM-specific gene expression such as croc-lacZ is deleted. From the restricted effects of ectopic byn /fkh, it has been proposed that the two genes are capable of specifying CVM development only in the region of high zfh-1 expression. zfh-1, byn and fkh act in parallel downstream of tll. High levels of caudal zfh-1, as with byn and fkh, are dependent on tll, and there is no crossregulation between zfh-1, byn and fkh (Kusch, 1999).

The Drosophila tracheal system is a model for the study of the mechanisms that guide cell migration. The general conclusion from many studies is that migration of tracheal cells relies on directional cues provided by nearby cells. However, very little is known about which paths are followed by the migrating tracheal cells and what kind of interactions they establish to move in the appropriate direction. An analysis has been carried out of how tracheal cells migrate relative to their surroundings and which tissues participate in tracheal cell migration. Cells in different branches are found exploit different strategies for their migration; while some migrate through preexisting grooves, others make their way through homogeneous cell populations. Alternative migratory pathways of tracheal cells are associated with distinct subsets of mesodermal cells and a model is proposed for the allocation of groups of tracheal cells to different branches. These results show how adjacent tissues influence morphogenesis of the tracheal system and offer a model for understanding how organ formation is determined by its genetic program and by the surrounding topological constraints (Franch-Marro, 2000).

Tracheal cells are first specified as clusters of ectodermal cells at the embryonic surface. Since tracheal cells invaginate and form the tracheal pits they occupy the grooves between the muscle precursors of adjacent metameres. The formation of this groove is independent of tracheal invagination because it also forms between metameres that do not have tracheal placodes and it also develops in trh mutant embryos, which do not undergo tracheal invagination. A subset of the tracheal cells moves anteriorly, whereas another subset moves posteriorly until they reach the cells from the adjacent placodes. These cells will form the dorsal trunk, the most prominent tracheal branch that spans the embryo longitudinally. Those cells migrate across the adjacent precursors of somatic muscles and separate the precursors of the most dorsal muscles from the precursors of more ventral muscles. Other cells, those from the dorsal side of the tracheal pit, move dorsally along the longitudinal groove to form the dorsal branches that will end up fusing with the dorsal branches coming from the contralateral hemisegments. In the ventral side, the tracheal cells follow two different paths along the two clusters of lateral muscle precursors at each side of the groove. Anterior ventral cells will form the anterior lateral trunk while the posterior ventral cells will form the posterior lateral trunk. Finally, another group of cells from a midposition in the tracheal pit will migrate inward and will form the visceral branch (Franch-Marro, 2000).

The migrating cells of the dorsal trunk do not recognize any preexisting gap between the muscle precursor cells. Thus, the role of the lateral mesoderm in the migration of tracheal cells was studied. The lateral mesoderm, which comprises the fat body and the somatic gonadal precursors, is determined by the early functions of tinman (tin) and zinc-finger homeodomain protein-1 (zfh1). In embryos mutant for either zfh-1 or tin, the number of somatic gonadal and fat body precursors is reduced. Consistent with a role for the lateral mesoderm in the migration of the dorsal trunk cells, it was observed that the development of the dorsal trunk is impaired in these mutants. This is a nonautonomous effect since tin and zfh-1 are not expressed in the tracheal cells. The effect is quite mild, however. Since tin and zfh-1 have overlapping and partially redundant functions, mutant embryos for both genes were examined. In those embryos, the somatic gonadal mesoderm and the fat body precursors are virtually absent and the tracheal dorsal trunk is almost completely absent, but formation of the dorsal and ventral branches is not impaired. This defect is not due to a failure of bnl expression since it appears at the right position between the tracheal pits. (Franch-Marro, 2000).

Zfh1, a somatic motor neuron transcription factor, regulates axon exit from the CNS

Motor neurons are defined by their axon projections, which exit the CNS to innervate somatic or visceral musculature, yet remarkably little is known about how motor axons are programmed to exit the CNS. This study describes the role of the Drosophila Zfh1 transcription factor in promoting axon exit from the CNS. Zfh1 is detected in all embryonic somatic motor neurons, glia associated with the CNS surface and motor axons, and one identified interneuron. In zfh1 mutants, ventral projecting motor axons often stall at the edge of the CNS, failing to enter the muscle field, despite having normal motor neuron identity. Conversely, ectopic Zfh1 induces a subset of interneurons -- all normally expressing two or more 'ventral motor neuron transcription factors' (e.g., Islet, Hb9, Nkx6, Lim3) -- to project laterally and exit the CNS. It is concluded that Zfh1 is required for ventral motor axon exit from the CNS (Layden, 2006).

Zfh1 is a transcriptional repressor required for mesoderm development and cell migration. This study shows that Zfh1 regulates lateral axon growth and CNS exit in ventral-projection motor neurons, and that misexpression of Zfh1 can induce lateral axon projections and an interneuron to motor neuron fate transformation in cells competent to respond to ectopic Zfh1. In vertebrates, there are typically two Zfh1 homologs: ZFHX1A and ZFHX1B in humans, deltaEF1 and Sip1 (also called ZEB1 and ZEB2, respectively) in mouse, and Kheper in fish. These Zfh1 orthologs are required for proper muscle and CNS development. The worm homolog zag-1 is expressed in muscle, interneurons, and motor neurons where it regulates axon outgrowth. Thus, the Zfh1 family members appear to have a conserved role regulating CNS and mesoderm development (Layden, 2006).

Drosophila Zfh1 is detected in all embryonic somatic motor neurons, but not in the dMP2 visceral motor neuron. Thus, its expression correlates better with lateral axon projection or innervation of body wall muscles rather than simply motor neuron identity. Zfh1 is detected in at least one interneuron that does not project laterally out of the CNS, the Apterous AD interneuron, and thus Zfh1 is not sufficient to induce lateral axon projections (except in certain contexts as described below). Zfh1 is also expressed in about 20 neurons that are negative for the motor neuron marker pMad and the glial marker Repo. These cells could be interneurons or late-differentiating motor neurons, but due to the lack of axon projection markers, it has not been possible to determine the identity of these cells. Zfh1 is expressed in a subset of glia, including the exit glia that enwrap motor nerves and the surface glia. The association of Zfh1⁺ exit glia with Zfh1⁺ motor neurons is intriguing; no phenotype is available that suggests a role for Zfh1 glial function (Layden, 2006).

Initially Zfh1 was investigated as a candidate for regulating the difference between motor neurons and interneurons. Analysis of the zfh1 mutant phenotype clearly shows that it is not required to promote motor neuron identity or suppress interneuron identity. All known motor neuron markers (Eve, Islet, Hb9, Lim3, Nkx6, Late bloomer, and pMad) and interneuron markers (Apterous, Engrailed, and Dachshund) are normal in zfh1 mutant and/or misexpression embryos (Layden, 2006).

Instead, it was found that zfh1 mutants show a severe defect in lateral axon projections of the ventral nerves ISNb/SNa; axons typically extend laterally to the edge of the CNS before arresting. This explains why there are missing nerves (the axons do not leave the CNS) but a normal pattern of nuclear pMad in motor neurons (they are at the edge of the CNS where they may be exposed to BMP signals from ventral muscles). In contrast, the dorsal projecting ISN appears to exit the CNS normally (100%), but is often truncated and has pathfinding defects. Although mesodermal defects cannot be rule out as the origin of these phenotypes, there are several reasons to think that the ISN defects are due to loss of neuronal Zfh1. (1) grain mutant embryos show completely normal muscle development but fail to express Zfh1 specifically in the aCC motor neuron, and the aCC shows truncation and pathfinding defects that are qualitatively and quantitatively similar to the zfh1 mutant ISN phenotype. (2) The grain mutant phenotype can be partially rescued by zfh1 expression specifically in the aCC motor neuron. Thus, Zfh1 does not appear to be required for ISN motor axon exit from the CNS, but rather for later aspects of motor axon outgrowth and target recognition (Layden, 2006).

The fact that ISNb and SNa motor axons stall at the edge of the CNS raises the possibility that the phenotype is due to loss of Zfh1 in the exit glia. Exit glia express Zfh1, are intimately associated with motor axons as they exit the CNS, and regulate the site of motor axon exit from the CNS. However, no glial positioning or migration phenotypes were observed, and the motor axon exit phenotype can be rescued with neuronal Zfh1 expression but not glial Zfh1 expression. Moreover, the lateral exit glia are not required for motor axons to exit the CNS; the only motor neuron known to require a non-neuronal guidance cue to exit the CNS is the transverse nerve motor neuron, which requires a specialized pair of midline mesodermal cells to exit the CNS. It is concluded that the zfh1 mutant motor neuron exit phenotype is due to loss of zfh1 in motor neurons (Layden, 2006).

In addition to Zfh1, two other Drosophila transcription factors regulate motor axon exit from the CNS. nkx6 mutants show a 90% loss of ISNb projection out of the CNS, despite normal Zfh1 levels in the affected neurons. Thus, both Nkx6 and Zfh1 are required for motor axon outgrowth via the ISNb nerve. Similarly, eve mutants show 98% and 80% loss of aCC and RP2 motor axon projections out of the ISN nerve, respectively, but in these mutants, aCC/RP2 neurons have little or no Zfh1 expression. Thus, it is unclear if Eve alone is required for ISN axon outgrowth, or if Eve and Zfh1 function redundantly to promote ISN axon outgrowth (Layden, 2006).

It has been reported that the expression of the Drosophila Eph transmembrane receptor is restricted to interneuronal axons, and that RNAi knockdown of Eph levels leads to redirection of some interneuron axons laterally out of the CNS. It is tempting to speculate that Zfh1 keeps Eph levels low in motor neurons to allow them to exit the CNS. However, the situation is complicated by the recent finding that the Eph null mutant does not show abnormal interneuron projections out of the CNS, and thus the role of Eph in restricting interneuron axons to the CNS remains unclear (Layden, 2006).

Zfh1 is likely to have additional later functions in motor neurons that have not been addressed. Vertebrate Zfh1 homologs ZEB-1 (Smad interacting protein 1/Sip1) and ZEB-2 (deltaEF1) are known to bind pSmad, the nuclear effecter of the BMP signaling pathway. In flies, BMP signaling from the muscle field induces nuclear pMad in motor neurons. This raises the possibility that Zfh1/pMad may collaborate to regulate later aspects of motor neuron differentiation. Analysis of later aspects of zfh1 mutant phenotype in motor neurons (such as target recognition, synaptic branching, or formation of a functional synapse) is complicated by the fact that zfh1 mutants have reduced muscle numbers and size. Analysis of potential later functions of Zfh1 awaits methods for removing zfh1 expression from individual motor neurons, similar to recent elegant studies of Eve motor neuron function (Layden, 2006).

Ectopic Zfh1 does not induce all neurons to project axons laterally out of the CNS, but rather a modest thickening of the ISNb nerve and a slight increase in the number of pMad⁺ neurons, due in part to the aberrant projection of dMP2 and the EW1-EW3 interneurons. Why do just these interneurons respond to ectopic Zfh1? The EW1-EW3 and dMP2 neurons are the only neurons that express two or more 'motor neuron transcription factors' (Hb9, Islet, Nkx6, or Lim3) but do not project to the lateral body wall muscles. However, the fact that dual misexpression of Zfh1/Hb9, Zfh1/Nkx6, or Hb9/Nkx6 does not induce a detectable increase in pMad staining or lateral projections above that observed in any of the single misexpression experiments suggests that the combined activity of these motor neuron factors is not sufficient to drive axon exit from the CNS. There may be an additional, unknown positive factor present in the EW1–EW3 and dMP2 neurons that gives them competence to exit the CNS in response to Zfh1 misexpression; alternatively, most interneurons could express a negative factor preventing lateral axon projection that cannot be overcome by Zfh1 expression (Layden, 2006).

How does Zfh1 induce nuclear pMad expression and lateral axon outgrowth in the EW1-EW3 and dMP2 neurons? In the EW1-EW3 neurons, expression of a constitutively-activated Tkv type I BMP receptor does not induce nuclear pMad; thus, misexpression of Zfh1 in EW1-EW3 must induce competence to respond to the BMP ligand as well as promote lateral axon outgrowth. In contrast, the dMP2 visceral motor neuron is competent to respond to BMP signaling even without endogenous Zfh1 expression. Thus, misexpression of Zfh1 may only promote lateral axon outgrowth in dMP2 (Layden, 2006).

The results support a role for Zfh1 in regulating lateral exit of the ISNb motor neurons from the CNS. Both zfh1 and nkx6 mutants show a loss of ISNb motor axons in the muscle field, suggesting that both genes are required to promote proper lateral CNS exit of ISNb motor neurons. Because Zfh1 and Nkx6 do not require each other for expression in ventral projecting motor neurons (e.g. RP1, 3, 4, 5), these two genes must be independently activated but are likely to have common downstream genes to induce lateral axon outgrowth. It will be interesting to determine if common targets of Zfh1 and Nkx6 are involved in promoting lateral CNS exit and if they are required for lateral CNS exit in other motor neuron subtypes. One might predict that a gene that regulates lateral CNS exit in all motor neurons would be a target of Zfh1, Nkx6, and Eve. One potential candidate is unc5, which encodes a repulsive Netrin receptor that promotes cell and growth cone movement away from Netrin on the midline. Misexpression of Unc5 within the Drosophila CNS results in a subset of interneurons redirecting their axon projections laterally out of the CNS. However, misexpression of Unc-5, but not Zfh1, results in lateral displacement of the EW1–EW3 cell bodies, so it is unlikely that the Zfh1 EW1–EW3 phenotype is solely due to upregulation of Unc-5 expression. In addition, zfh1 mutant ISNb motor neurons initially project away from the midline, only stalling at the edge of the CNS, which is inconsistent with the phenotype being due to failure to express the Unc-5 receptor. In the future, it will be important to test directly whether Zfh1 regulates unc-5 directly or indirectly, as well as to identify novel direct targets regulated by Zfh1. Because eve and nkx6 mutants also show motor neuron axon exit defects, it will be especially interesting to compare direct targets of Eve, Nkx6, and Zfh1; common targets may help identify genes important for promoting motor neuron guidance out of the CNS in both flies and vertebrates (Layden, 2006).

Functional conservation of zinc-finger homeodomain gene zfh1/SIP1 in Drosophila heart development

Comparative genetic studies of diverse animal model systems have revealed that similar developmental mechanisms operate across the Metazoa. In many cases, the genes from one organism can functionally replace homologues in other phyla, a result consistent with a high degree of evolutionarily conserved gene function. Functional conservation was investigated between the Drosophila Zinc-finger homeodomain protein 1 (Zfh1) and its mouse functional homologue Smad-interacting protein 1 (SIP1). Northern blot analyses of SIP1 expression patterns detected three novel variants (8.3, 2.7, and 1.9 kb) in addition to the previously described 5.3 kb SIP1 transcript. The two shorter novel SIP1 transcripts were encountered only in developing embryos and both lacked zinc-finger clusters or homeodomain regions. The SIP1 transcripts showed complex embryonic expression patterns consistent with that observed for Drosophila Zfh1. They were highly expressed in the developing nervous systems and in a number of mesoderm-derived tissues including lungs, heart, developing myotomes, skeletal muscle, and visceral smooth muscle. The expression of the mammalian 5.3 kb SIP1 transcript in Drosophila zfh1 null mutant embryos completely restored normal heart development in the fly, demonstrating their functional equivalence in cardiogenic pathways. These data, together with the previously described heart defects associated with both SIP1 and Drosophila zfh1 mutations, solidify the conclusion that the Zfh1 family members participate in an evolutionary conserved program of metazoan cardiogenesis (Liu, 2006).

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date revised: 15 July 2008

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